The present invention relates to an apparatus and a method for performing automated amplification of nucleic acids under standardized conditions. The invention also relates to a disposable part for the apparatus used in the method.
The amplification of nucleic acids is described in U.S. Pat. No. 4,683,194, U.S. Pat. No. 4,683,195 and U.S. Pat. No. 4,683,202, and in patent applications EP 200 362, EP 229 701, EP 237 362 and U.S. 024 604. In amplification, a reaction mixture which contains the single-stranded target nucleic acid, at least two suitable primers, four different deoxyribonucleoside triphosphates, and DNA polymerase in a suitable buffer solution is first incubated at a suitable temperature in order to polymerize the DNA. Thereafter the double-stranded DNA formed in the polymerization is denatured by heating and the reaction mixture is cooled to a temperature at which the primers are capable of again hybridizing with the target DNA. When necessary, the temperature of the reaction mixture is adjusted to a temperature optimal for the action of DNA polymerase, and DNA polymerase is added. The steps described above are repeated as many times as is necessary for producing the desired result.
Amplification has in general been performed manually by transferring the test tubes from one place to another dozens of times. The method is slow and cumbersome to perform. When thermolabile polymerase has been used, it has been necessary to open and close the tubes at intervals. Furthermore, in order to produce a homogenous reaction fluid, it has been necessary to centrifuge from the walls of the tubes the reaction fluid condensed during the cooling step. It has been a further disadvantage that the reaction conditions vary during repeated steps. This has constituted a problem, especially when a thermostable enzyme has been used, since in spite of its thermostability the polymerase is destroyed if it is kept at a denaturation temperature for too long.
Patent application EP 236 069 describes an apparatus in which the amplification of nucleic acids is performed under computer control by heating and cooling the reaction mixture in the same vessel. This apparatus has certain disadvantages. It is difficult to heat and cool a reaction mixture with precision and with sufficient speed, since in addition to the temperature of the vessel and the reaction mixture in it, the temperature of the surrounding apparatus also has to be adjusted. The heat capacity of the surrounding apparatus is inevitably considerable compared with the heat capacity of the small-volume (approximately 100 .mu.l) reaction mixture. For this reason, the reaction mixture is at a temperature which is disadvantageous for amplification for a significant portion of each cycle. Problems arise in particular in the controlling of the denaturation temperature and time. If the temperature is too low, denaturation will not proceed in the manner desirable with respect to amplification. On the other hand, if the polymerase has to be at or near the denaturation temperature for too long, even thermally stable polymerases will be destroyed. Thus, in practice, only a thermostable polymerase can be used and it is active for the duration of only a few reaction cycles.
For the reasons stated above it has not been possible to fully automate DNA amplification under standardized optimal conditions. The only automated system to date (described in EP 236,069) requires the use of a thermostable DNA polymerase and does not function under optimal conditions.
The object of the present invention is to provide an apparatus and method eliminating the above-mentioned disadvantages. By using the apparatus of the present invention it is possible to fully automate the amplification of nucleic acids in one or several samples simultaneously under standardized optimal reaction conditions. In the apparatus according to the invention, the correct denaturation temperature and time can be adjusted with sufficient precision and speed so as not to significantly denature thermally stable DNA polymerase. The method and apparatus according to the invention can be used for the amplification of nucleic acids regardless of whether the polymerase used is thermostable or not.